Target details

Target details

Autophagy is a process of intracellular bulk degradation in which cytoplasmiccomponents, including organelles, are sequestered within double-membrane vesiclesthat deliver the contents to the lysosome/vacuole for degradation. Duringmacroautophagy, the sequestering vesicles, termed autophagosomes, fuse with thelysosome or vacuole resulting in the delivery of an inner vesicle (autophagic body) intothe lumen of the degradative compartment. There are 16 proteins participating in theautophagy pathway in human. The autophagy protein LC3, a mammalian homologue ofAtg8, was originally identified as microtubule-associated protein 1 light chain 3. It is acomponent of both the MAP1A and MAP1B microtubule-binding domains and theheavy-chain independent regulation of LC3 expression might modify MAP1microtubule-binding activity during development. LC3 is the only known mammalianprotein identified that stably associates with the autophagosome membranes. LC3-I iscytosolic and LC3-II is membrane bound and enriched in the autophagic vacuolefraction. The detection of LC3-I to LC3-II conversion is a useful and sensitive marker fordistinguishing autophagy in mammalian cells. Alternate Names: anti-Microtubule-associated proteins 1A/1B light chain 3B MAP1A/MAP1B LC3 Bantibody, anti-MAP1A/b light chain 3 B antibody, anti-Autophagy-related protein LC3 Bantibody, anti-Autophagy-related ubiquitin-like modifier LC3 B antibody.Gene Symbol: MAP1LC3A

Western Blot protocol specific for LC3 Antibody Western Blot Protocol1. Perform SDS-PAGE (4-12 %) on samples to be analyzed, loading 40 µg of total protein per lane.. Transfer proteins to Nitrocellulose according to the instructions provided by the manufacturer of the transfer apparatus.. Rinse membrane with dH2O and then stain the blot using ponceau S for 1-2 minutes to access the transfer of proteins onto the nitrocellulose membrane. Rinse the blot in water to remove excess stain and mark the lane locations and locations of molecular weight markers using a pencil.. Rinse the blot in TBS for approximately 5 minutes.. Block the membrane using 5 % non-fat dry milk + 1 % BSA in TBS for 1 hour at room temperature (RT).. Rinse the membrane in dH2O and then wash the membrane in wash buffer [TBS + 0.1 % Tween] 3 times for 10 minutes each.. Dilute the rabbit anti-LC3 primary antibody in blocking buffer and incubate 1 hour at RT.. Rinse the membrane in dH2O and then wash the membrane in wash buffer [TBS + 0.1 % Tween] 3 times for 10 minutes each.. Apply the diluted rabbit-IgG HRP-conjugated secondary antibody in blocking buffer (as per manufacturer's instructions) and incubate 1 hour at room temperature.. Wash the blot in wash buffer [TBS + 0.1 % Tween] 3 times for 10 minutes each (this step can be repeated as required to reduce background).. Apply the detection reagent of choice in accordance with the manufacturers instructions (we used BioFX Super Plus ECL). Note: Tween-20 can be added to the blocking or antibody dilution buffer at a final concentration of 0.05-0.2 %, provided it does not interfere with antibody-antigen binding.I. Deparaffinization: A. Treat slides with Xylene: 3 changes for 5 minutes each. Drain slides for 10 seconds between changes.B. Treat slides with 100 % Reagent Alcohol: 3 changes for 5 minutes each. Drain slides for 10 seconds between changes.II. Quench Endogenous Peroxidase: To Prepare 200 mL of Quenching Solution: Add 3 mL of 30 % Hydrogen Peroxide to 200 mL of Methanol.**Use within 4 hours of preparationA.Place slides in peroxidase quenching solution: 15-30 minutes.III. Retrieve Epitopes: A. Preheat Citrate

Lee, Cao, Mostoslavsky et al.: "A role for the NAD-dependent deacetylase Sirt1 in the regulation of autophagy." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 105, Issue 9, pp. 3374-9, 2008 (PubMed).

Zhang, Yu, Pan et al.: "Small molecule regulators of autophagy identified by an image-based high-throughput screen." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 104, Issue 48, pp. 19023-8, 2007 (PubMed).